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Characterization of Monoclonal Antibodies to Junin Virus Nucleocapsid protein 1
and Application to the Diagnosis of Hemorrhagic Fever Caused by South 2
American Arenaviruses 3
Mina Nakauchi1, Shuetsu Fukushi
1, Masayuki Saijo
1, Tetsuya Mizutani
1, Agustín E. 4
Ure2, Victor Romanowski
2, Ichiro Kurane
1, and Shigeru Morikawa
1 5
1 Department of Virology 1, National Institute of Infectious Diseases, 4-7-1 Gakuen, 6
Musashimurayama, Tokyo 208-0011, Japan, and 2 Facultad de Ciencias Exactas, 7
Instituto de Biotecnología y Biología Molecular, Universidad Nacional de La Plata, 8
Consejo Nacional de Investigaciones Científicas y Técnicas 9
(IBBM-UNLP-CONICET-CCT La Plata), La Plata, Argentina 10
11
Running title: Antigen capture ELISA for South American Arenaviruses 12
13
Corresponding author: Shigeru Morikawa, Ph.D., D.V.M., Department of Virology 1, 14
National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 15
208-0011, Japan, Tel: +44-42-561-0771; Fax: +44-42-561-2039; E-mail: 16
18
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00163-09 CVI Accepts, published online ahead of print on 24 June 2009
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Abstract 1
The Junin virus (JUNV), Machupo virus, Guanarito virus, Sabia virus, and Chapare 2
virus, are members of the Clade B New World arenaviruses and are the etiological 3
agents of viral hemorrhagic fevers that occur in South America. In this study, we 4
produced three monoclonal antibodies (MAbs) to recombinant nucleocapsid protein of 5
JUNV, designated as MAbs C6-9, C11-12, and E4-2. The specificity of these MAbs was 6
examined by enzyme-linked immunosorbent assay (ELISA), indirect 7
immunofluorescence assay, and an epitope mapping method. Using these MAbs, we 8
developed antigen (Ag)-capture ELISA systems. We showed that, by using MAb C6-9, 9
JUNV antigen was specifically detected. On the other hand, by using MAb C11-12 or 10
E-4-2, the antigens of all human pathogenic South American arenaviruses were detected. 11
The combined use of these Ag-capture ELISA systems in the present study may be 12
useful for the diagnosis of acute phase viral hemorrhagic fever due to infection by a 13
South American arenavirus. 14
15
16
17
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Introduction 1
The South American arenaviruses Junin virus (JUNV), Machupo virus (MACV), 2
Guanarito virus (GTOV), Sabia virus (SABV), and Chapare virus (CHPV) are members 3
of the Clade B New World arenaviruses. JUNV, MACV, GTOV, and SABV are the 4
etiological agents of Argentine hemorrhagic fever (AHF), Bolivian hemorrhagic fever 5
(BHF), Venezuelan hemorrhagic fever (VHF), and Brazilian hemorrhagic fever, 6
respectively (4). CHPV was also recently shown to be associated with cases of 7
hemorrhagic fever in Bolivia (5). AHF emerged in the 1950s, and since then, outbreaks 8
have occurred annually without interruption (4). The mortality rate for AHF is estimated 9
to be 15-30%, but early treatment with immune plasma reduces the rate to less than 1% 10
(6). The region at risk has been progressively expanding into North-central Argentina, 11
and almost 5 million people are currently considered to be at risk for AHF (6, 13). 12
Phylogenetic analysis indicates that JUNV is more closely related to MACV than to 13
SABV or CHPV, whereas SABV and CHPV are more closely related to each other than 14
to other New World arenaviruses (5). 15
Arenaviruses are enveloped and contain a bisegmented RNA genome. The genome 16
consists of two ambisense single-stranded RNA molecules, one designated L, which 17
encodes the RNA-dependent RNA polymerase and a zinc-binding matrix protein Z, and 18
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the other designated S, which encodes the major structural components of the virion: the 1
nucleocapsid protein (NP) and the envelope glycoprotein precursor (GPC) (15). The 2
arenavirus NP is the most abundant protein among the viral structural proteins both in 3
infected cells and in virions (2) and is commonly used as a target for detecting viral 4
antigens (20). Moreover, arenavirus NPs have been known to be the most conserved 5
among the same virus species and, to some extent, among different arenavirus species 6
(3, 8). Therefore, it seems likely that monoclonal antibodies (MAbs) raised against the 7
NP of an arenavirus would also be useful for detecting other arenaviruses (20). Recently, 8
an immunoglobulin G (IgG)-enzyme linked immunosorbent assay (ELISA) was 9
developed using a recombinant NP (rNP) of JUNV, obtained from a recombinant 10
baculovirus system, and was proposed to be useful for etiologic confirmation of AHF in 11
seroepidemiological studies (20, 26). It is considered that an antigen (Ag)-capture 12
ELISA using MAbs specific for viral antigens allows rapid diagnosis of the acute phase 13
of viral HF by detecting viral antigens in blood or tissue homogenates (20). In this study, 14
we produced MAbs to rNP of JUNV. These MAbs were characterized by ELISA, 15
indirect immunofluorescence assay (IFA), and an epitope mapping method. Ag-capture 16
ELISAs were developed using these MAbs that are specific for JUNV and that are 17
broadly applicable for the detection of human pathogenic New World arenaviruses. 18
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1
Materials and Methods 2
Cell culture 3
Hybridomas and their parental cell line, P3/Ag568, were maintained in RPMI 1640 4
(Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine 5
serum (FBS), nonessential amino acids (Invitrogen), and antibiotics (streptomycin and 6
penicillin G, Invitrogen). Hypoxanthine-aminopterin-thymidine supplement (Invitrogen) 7
was added to the medium for selection of hybridomas, as recommended by the supplier. 8
BTI-TN-5B1-4 (High FiveTM
, Invitrogen) insect cells were maintained in TC100 9
(Invitrogen) supplemented with 10% FBS, 2% tryptose phosphate broth (Difco, Detroit, 10
MI), and kanamycin (Invitrogen). HeLa cells were maintained in minimal essential 11
medium (MEM) (Sigma-Aldrich, St. Louis, MO) supplemented with 5% FBS and 12
antibiotics (streptomycin and penicillin G, Invitrogen). 13
14
Recombinant baculoviruses 15
The baculoviruses Ac-JUNV-NP and Ac-His-Lassa virus (LASV)-NP, expressing 16
JUNV-rNP and His-LASV-rNP, respectively, were generated as described previously 17
(20). 18
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The cDNAs of MACV, GTOV, SABV, and CHPV NP were obtained by chemical 1
synthesis (Codon Devices, Cambridge MA). The GenBank accession numbers of the 2
nucleotide sequences of the MACV, GTOV, SABV, and CHPV NP genes are 3
NC_005078, AF485258, NC_006317, and NC_010562, respectively. The cDNAs of 4
MACV, GTOV, SABV, and CHPV NP were digested with BamHI and subcloned into 5
the BamHI restriction site of pAcYM1 (14), and designated as pAcYM1-MACV-NP, 6
pAcYM1-GTOV-NP, pAcYM1-SABV-NP, and pAcYM1-CHPV-NP, respectively. High 7
FiveTM
cells were transfected with mixtures of linearized BacPAK6 DNA (Clontech, 8
Mountain View CA) and the recombinant transfer vector according to the 9
manufacturer’s instructions and the procedures described by Kitts et al. (10), from 10
which recombinant baculoviruses were obtained. The baculoviruses expressing 11
MACV-rNP, GTOV-rNP, SABV-rNP, and CHPV-rNP were designated as Ac-MACV-NP, 12
Ac-GTOV-NP, Ac-SABV-NP, and Ac-CHPV-NP, respectively. 13
14
Expression and purification of rNP 15
High FiveTM
cells infected with Ac-JUNV-NP, Ac-MACV-NP, Ac-GTOV-NP, 16
Ac-SABV-NP, Ac-CHPV-NP, or Ac-His-LASV-NP were incubated at 26 °C for 72 h. 17
The cells were then washed twice with cold phosphate-buffered saline (PBS) solution. 18
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The High FiveTM
cells were lyzed in PBS containing 1% NP40 and 2 M urea. After 1
centrifuging the cell lysates at 15,000 x g for 10 min, the pellet fractions were collected 2
and then solubilized in PBS containing 8 M urea. After the samples were centrifuged, 3
the supernatant fractions were used as the purified antigens. The control
antigen was 4
produced from High FiveTM
cells infected with Ac-∆P, which lacks the polyhedrin gene, 5
in the same manner as that for the negative control antigens. All antigens were aliquoted 6
and kept at -80 °C until use. 7
8
Establishment of monoclonal antibodies 9
BALB/c mice were immunized three times with the purified JUNV-rNP. Spleen cells 10
were obtained 3 days after the last immunization and fused with P3/Ag568 cells using 11
polyethylene glycol (Invitrogen). The culture supernatants of the hybridoma cells were 12
screened by ELISA with purified JUNV-rNP as an antigen in the presence of 2 M urea. 13
MAbs were purified from the culture supernatant using a MAbTrap GII antibody 14
purification kit (GE Healthcare Bio-Sciences, Piscataway, NJ) according to the 15
manufacturer’s instructions. The concentration of each purified MAb was also 16
determined by the Bradford method using Protein Assay (Bio-Rad Laboratories, 17
Hercules CA) according to the manufacturer’s instructions. 18
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1
Polyclonal antibodies 2
Polyclonal antibodies were induced in rabbits by immunization with the purified rNP of 3
JUNV, MACV, GTOV, SABV, and CHPV, respectively. Rabbit sera collected before 4
immunization were used as controls. 5
6
IgG-ELISA 7
IgG-ELISA was performed as previously described except for the antigen preparation 8
(20-22). Briefly, ELISA plates (96-well plate, Pro-Bind; Falcon; Becton Dickinson
9
Labware, Franklin Lakes, NJ) were coated with the predetermined optimal quantity of 10
purified JUNV-rNP, MACV-rNP, GTOV-rNP, SABV-rNP, CHPV-rNP, or 11
His-LASV-rNP (approximately 100 ng/well) at 4 °C overnight. Then, each well of the 12
plates was covered with 200 µl of PBS containing 5% skim milk and 0.05% Tween 20 13
(PBST-M), followed by incubation for 1 h for blocking at 37ºC. The plates were washed 14
three times with PBS containing 0.05% Tween-20 (PBST) and then inoculated with 15
MAbs (100 µl/well), which were diluted 1:1,000 with PBST-M.
After a 1 h incubation 16
period, the plates were washed three times with PBST, and then the plates were 17
inoculated with goat anti-mouse IgG antibody labeled with horseradish peroxidase 18
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(HRP) (1:1,000 dilution; Zymed Laboratory, Inc., South San Francisco, CA). After a 1
further 1 h incubation period, the plates were washed, and 100 µl of ABTS 2
[2,2'azinobis(3-ethylbenzthiazolinesulfonic acid)] solution (Roche Diagnostics, 3
Mannheim, Germany) were added to each well. The plates were incubated for 30 min at 4
room temperature, and the optical density at 405 nm (OD405) was measured against
a 5
reference of 490 nm. The adjusted OD405 value was calculated by subtracting the OD405 6
value of the negative antigen-coated wells from that of the corresponding wells. 7
8
Indirect immunofluorescence assay 9
A full-length cDNA of JUNV NP obtained from Ac-JUNV-NP, which possessed a 10
BamHI restriction site at both extremities, was cloned into the BamHI site of the 11
pKS336 vector (23), and designated as pKS-JUNV-NP. Also, chemically synthesized 12
full-length cDNAs of MACV NP, GTOV NP, SABV NP, and CHPV NP were cloned 13
into the BamHI site of the pKS336 vector, and designated as pKS-MACV-NP, 14
pKS-GTOV-NP, pKS-SABV-NP, and pKS-CHPV-NP, respectively. HeLa cells were 15
then transfected with each of these expression plasmids by using a transfection reagent 16
(FuGENE6, Roche Diagnostics) according to the manufacturer's instructions. The
17
transfected cells were selected with 4 µg of blasticidin S-hydrochloride/ml in culture 18
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medium. The HeLa cell clones were obtained by analyzing expression of each rNP by 1
indirect-immunofluorescence using rabbit serum raised against JUNV-rNP, MACV-rNP, 2
GTOV-rNP, SABV-rNP, or CHPV-rNP, as previously described (20). 3
4
Ag-capture ELISA 5
Purified MAb C6-9, C11-12, or E4-2 was coated onto microwell immunoplates (Falcon, 6
Becton Dickinson Labware) at >100 ng/well in 100 µl of PBS at 4 oC overnight, 7
followed by blocking with PBST-M for 1 h at room temperature (RT). After the plates 8
were washed with PBST, 100 µl of samples containing serially diluted rNP of JUNV, 9
MACV, GTOV, SABV, CHPV, or LASV were added, and the plates were incubated for 10
1 h at 37 oC. The plates were then washed with PBST, and 100 µl of rabbit polyclonal 11
antibody raised against rNP of JUNV diluted 1:500 with PBST-M was added to each 12
well. After 1 h incubation at 37 oC, the plates were washed with PBST, and 13
HRP-conjugated goat anti-rabbit IgG (Zymed, San Francisco CA) was added. The 14
plates were incubated for 1 h at RT. After another extensive wash with PBST, 100 µl of 15
ABTS substrate solution (Roche Diagnostics) were added, and the OD was measured at 16
a wavelength of 405 nm with a reference wavelength 490 nm after 30 min of incubation 17
at RT. As a negative control, the OD of control antigen inoculated wells was measured. 18
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The adjusted OD405 values were calculated by subtracting the OD405 value of the 1
negative control well from the corresponding OD405 values. Mean and standard 2
deviation were calculated from the ODs of 12 negative control wells and the cutoff 3
value for the assay was defined as the mean plus 3 standard deviations. 4
5
Expression of truncated rNPs of JUNV 6
In order to determine the epitope on the JUNV-rNP by the MAbs, a series of
truncated 7
JUNV-rNPs were expressed as fusion proteins with glutathione S-transferase (GST). 8
The DNA corresponding to each of the truncated NP fragments was
amplified by PCR 9
using specifically designed primer sets. The amplified DNA was subcloned into the 10
BamHI and EcoRI cloning sites of plasmid pGEX-2T (Amersham Pharmacia Biotech, 11
Buckinghamshire, England). The GST-tagged full-length rNP (GST- JUNV-frNP) or 12
truncated forms of rNP (GST- JUNV-trNP) were expressed in Escherichia coli BL21 13
and then partially purified. 14
15
Western Blotting 16
The MAbs were tested for reactivity to GST-JUNV-frNP and a series of 17
GST-JUNV-trNPs by Western blotting as reported previously (9, 18, 24). 18
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1
Epitope mapping of MAbs 2
For MAbs C6-9 and C11-12, epitopes were determined by epitope blocking ELISA 3
using synthetic peptides. The deca-peptides were chemically synthesized by shifting 4
one amino acid (aa), with a consecutive overlap of 9 aa to cover the JUNV-NP (aa 5
positions 5-26 for C6-9 and aa positions 543-564 for C11-12). ELISA plates were 6
coated with purified JUNV-rNP prepared using a baculovirus expression system 7
(approximately 100 ng/well) at 4 °C overnight. Then, each well of the plates was 8
inoculated with 200 µl of PBS-M, followed by incubation for 1 h for blocking. MAb 9
C6-9 or C-11-12 was mixed with each peptide (1 µg/well) and incubated for 1 h at 37 10
oC, and then the mixture was added to each well of the plates. After a 1 h incubation 11
period, the plates were washed three times with PBST, and then the plates were 12
inoculated with goat anti-mouse IgG antibody labeled with HRP (1:1,000 dilution;
13
Zymed). The following procedure was performed as described in the IgG-ELISA 14
section above. 15
For MAb E4-2, the epitope was determined by ELISA using GST-JUNV-frNP and 16
-trNPs. ELISA plates were coated with purified GST-JUNV-frNP and -trNPs 17
(approximately 100 ng/well) according to the method described in the IgG-ELISA 18
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section above. MAb E4-2 or anti-GST MAb was used for detection at 1:2,000 or 1:500 1
dilutions, respectively. The adjusted OD405 was calculated by dividing the OD405 of 2
MAb E4-2 by that of anti-GST MAb from the corresponding wells. 3
4
Results 5
Generation of monoclonal antibodies 6
In order to obtain MAbs against JUNV-NP, BALB/c mice were immunized with the 7
purified rNP of JUNV. The MAbs were purified and tested for reactivity to the rNP of 8
JUNV by IgG-ELISA. Three MAbs, designated as MAb C6-9, MAb C11-12, and MAb 9
E4-2, reacted with the rNP of JUNV by IgG-ELISA even in the presence of 2 M urea. 10
11
Reactivity of MAbs to rNP of arenaviruses 12
Reactivity of MAbs with rNPs of human pathogenic arenaviruses was examined using 13
ELISA. MAb C6-9 reacted specifically with rNP of JUNV, but did not react with those 14
of the other pathogenic South American arenaviruses (Fig. 1). On the other hand, 15
C11-12 reacted at the same level with rNP of all pathogenic South American 16
arenaviruses including JUNV, GTOV, MACV, SABV, and CHPV. MAb E4-2 reacted 17
strongly with rNP of JUNV, slightly weakly with GTOV, MACV and SABV, and very 18
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weakly with CHPV. However, MAb E4-2 reacted clearly with rNP of CHPV when the 1
wells of ELISA plate were coated with more concentrated CHPV antigen (data not 2
shown). None of the three MAbs reacted with rNP of the human pathogenic Old World 3
arenavirus, LASV. 4
Reactivity was also examined by IFA. Consistent with the result by ELISA, MAb C6-9 5
reacted only with HeLa cells expressing rNP of JUNV, and MAb C11-12 reacted with 6
HeLa cells expressing rNPs of all pathogenic South American arenaviruses (Table 1). 7
On the other hand, MAb E4-2, which showed cross-reactivity to other arenaviruses by 8
ELISA, reacted only with HeLa cells expressing rNP of JUNV (Table 1). None of the 9
three MAbs reacted with LASV NP-expressing HeLa cells (Table 1). 10
11
Development of Ag-capture ELISA 12
Ag-capture ELISAs were developed using three MAbs as capture antibodies, and the 13
sensitivity and specificity were determined. The Ag-capture ELISA with MAb C6-9 14
specifically detected rNP of JUNV, whereas it could not detect rNPs of the other South 15
American arenaviruses. No less than 62.5 ng/well of rNP of JUNV were detected by 16
Ag-capture ELISA using MAb C6-9 (Fig. 2A). On the other hand, Ag-capture ELISA 17
using MAb C11-12 and E4-2 was more sensitive at detecting rNP of JUNV with 18
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detection limits of 7.82 and 3.91 ng/well, respectively, and these Ag-capture ELISAs 1
also detected rNPs of the other South American arenaviruses GTOV, MACV, SABV, 2
and CHPV (Fig. 2B, C). In contrast, LASV-NP was not detected by any of the 3
Ag-capture ELISAs. 4
5
Determination of the epitope on JUNV-rNP recognized by the MAbs 6
In order to determine regions including epitopes on the JUNV-rNP recognized by the 7
MAbs, reactivity of these MAbs was tested by Western blotting using GST-JUNV-frNP 8
and a series of GST-JUNV-trNPs as antigens. The MAb C6-9 reacted with 9
GST-JUNV-frNP and GST-JUNV-trNPs at aa positions 384-564 and 524-564 (Fig. 3). 10
The MAb C11-12 reacted with GST- JUNV-frNP and GST-JUNV-trNPs at aa positions 11
1-235 and 1-59 (Fig. 3). The MAb E4-2 reacted with GST- JUNV-frNP and 12
GST-JUNV-trNPs at aa positions 1-235 and 54-117 (Fig. 3). 13
To further determine exact epitope positions on rNP of JUNV, we performed epitope 14
blocking ELISA using a series of overlapping synthetic peptides. As shown in Fig. 4A, 15
peptides containing PPSLLFLP (aa positions 551-558) blocked the reaction of MAb 16
C6-9 with the purified rNP of JUNV. Similarly, peptides containing WTQSLR (aa 17
positions 12-17) blocked the reaction of MAb C11-12 with the purified rNP of JUNV 18
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(Fig. 4B). 1
Because the epitope recognized by MAb E4-2 could not be determined by epitope 2
blocking ELISA, that was analyzed more in detail using a series of GST-JUNV-trNP by 3
ELISA (Fig. 4C). The reactivity of MAb E4-2 was normalized by dividing the OD405 4
value of MAb E4-2 by that of anti-GST MAb. MAb E4-2 reacted with the 5
GST-JUNV-trNP containing the polypeptides, KEVDRLMS (aa positions 72-79). The 6
result of ELISA was consistent with that of Western blotting (data not shown). Epitopes 7
recognized by the MAbs are summarized in Fig. 5. 8
9
Discussion 10
Detection of viral antigen and/or genome is crucial for rapid diagnosis of patients with 11
HF caused by South American arenaviruses, especially for patients in acute phase. The 12
application of reverse transcriptase polymerase chain reaction (RT-PCR) and TaqMan 13
PCR for detection of JUNV, MACV, and GTOV genome has been reported (1, 11, 12, 14
27). Serological diagnosis is also useful for the diagnosis of AHF especially for patients 15
in convalescent phase (7, 17, 19, 20, 26). 16
Ag-capture ELISA using a cocktail of MAbs against JUNV (25) was applied in an 17
epidemiological study of rodents in Argentina (16). MAbs reactive with NP of JUNV 18
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have been shown to cross-react with those of MACV and other non-pathogenic 1
arenaviruses (25). In the present study, by using MAbs raised against rNP of JUNV, we 2
developed an Ag-capture ELISA specific for JUNV and those broadly reactive to human 3
pathogenic New World arenaviruses. 4
The three MAbs to JUNV-NP (designated as MAb C6-9, MAb C11-12, and MAb E4-2) 5
reacted with rNP of JUNV prepared using a baculovirus expression system by 6
IgG-ELISA and with rNP expressed in mammalian cells by IFA (Fig. 1 and Table 1). All 7
Ag-capture ELISAs using MAbs E4-2, C11-12, and C6-9 detected rNP of JUNV (Fig. 8
2), suggesting that these ELISAs are a useful tool for the diagnosis of AHF. 9
Interestingly, Ag-capture ELISA using MAb E4-2 detected the antigen of all pathogenic 10
South American arenaviruses tested in addition to that of JUNV (Fig. 2). IgG-ELISA 11
showed that the reactivity of MAb E4-2 with rNP of JUNV was stronger than that with 12
rNPs of other South American arenaviruses (Fig. 1). A minimal length of the epitope 13
required to be recognized by MAb E4-2 was some 8 amino acids with a sequence of 14
KEVDRLMS (Fig. 4 and 5). However, GST-JUNV-trNP at aa positions 1-80 was more 15
reactive compared with that at aa positions 1-79 which includes minimal epitope 16
sequences, but was still less reactive than those at aa positions 72-564, 67-564 and 17
1-564 (Fig. 4). Even though we could not express GST-JUNV-trNPs at aa positions 1-81 18
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or more in E. coli because of their toxicity, it might be possible that some additional 1
amino acids at the C-terminus of the minimal epitope are required for complete reaction 2
with MAb E4-2. Actually, comparison of amino acid sequences of NPs at positions 72 3
-83 among South American arenaviruses showed that differences in amino acids with 4
respect to JUNV were 1 amino acid for GTOV, 2 for MACV, 3 for SABV and 5 for 5
CHPV (Fig. 5), and these differences were correlated well with levels of reactivity of 6
MAb E4-2 to rNPs of each viruses (Fig. 1). 7
Ag-capture ELISA using MAb C11-12 also detected the antigen of all other pathogenic 8
South American arenaviruses (Fig. 2). The MAb C11-12 reacted with rNP of all 9
pathogenic South American arenaviruses by IgG-ELISA and IFA (Fig. 1 and Table 1). 10
These results suggest that the MAb C11-12 would be useful for detecting the antigen of 11
all South American arenaviruses by Ag-capture ELISA and IFA. Furthermore, the aa 12
sequence (WTQSLR) of the epitope recognized by MAb C11-12 was located at the 13
N-terminus of the JUNV-NP and was conserved among all pathogenic South American 14
arenavirus isolates so far deposited in GenBank (Fig. 5). However, slight differences in 15
sensitivity of detection of the NPs of each South American arenavirus by Ag-capture 16
ELISA were observed. This may be due to the reactivity of the detector antibody, 17
anti-JUNV-NP rabbit serum, which was raised against the purified rNP of JUNV. Since 18
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the N-terminal regions of NPs recognized by C11-12 and E4-2 (aa positions 1-80) were 1
relatively conserved among the NPs of South American arenaviruses, Ag-capture 2
ELISAs using MAbs C11-12 and E4-2 are considered to be useful for detecting most 3
South American arenavirus isolates. Therefore, these Ag-capture ELISAs may not only 4
be applicable for the diagnosis of AHF, but also for the diagnosis of BHF, VHF, and 5
Brazilian HF, and may also be applicable for newly emerging viral HFs caused by 6
CHPV, although further study is needed. 7
On the other hand, Ag-capture ELISA using MAb C6-9 only detected JUNV antigen 8
(Fig. 2). Furthermore, MAb C6-9 only reacted with rNP of JUNV by IgG-ELISA and 9
IFA (Fig. 1 and Table 1). The aa sequence (PPSLLFLP) of the epitope recognized by 10
MAb C6-9 was conserved among JUNV isolates so far deposited in GenBank (data not 11
shown), but differed from other South American arenavirus isolates (Fig. 5). Since only 12
proline at aa position 552 in the epitope sequences is different in MACV, this proline is 13
likely to be critical in reaction of MAb C6-9. Therefore, Ag-capture ELISA using MAb 14
C6-9 may detect most if not all JUNV isolates. Considering that the symptoms due to 15
JUNV infection in humans are indistinguishable from those due to other South 16
American arenaviruses, Ag-capture ELISA using MAb C6-9 may be a useful diagnostic 17
tool especially for AHF. 18
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While the efficacy of newly developed Ag-capture ELISAs in the diagnosis
of viral HF 1
caused by South American arenaviruses was not evaluated using serum samples from 2
patients, it is generally accepted that Ag-capture ELISA is useful for the detection of 3
viral antigens in blood and/or other organ tissue specimens from patients in acute phase. 4
The aa sequences of the epitope regions recognized by MAb E4-2 and C6-9 were 5
different from those of the corresponding region of LASV. On the other hand, the aa 6
sequence (WTQSLR) of the epitope recognized by MAb C11-12 is identical between 7
JUNV and LASV, even though the MAb failed to react to LASV-NP. However, the 8
amino acid residue at position 8 and 11 are proline and arginine in the NP of South 9
American arenaviruses, while they are lysine / arginine and leucine in LASV-NP, 10
respectively. Thus, it might be possible that some differences in aa sequences around the 11
minimal epitope region affect the reaction with MAb C11-12. 12
In general, RT-PCR is more sensitive in detecting viruses in patients’ specimens 13
compared with Ag-capture ELISA. Recently, real time RT-PCR has been established for 14
the detection of all pathogenic South American arenaviruses, but it has not yet been 15
applied for clinical specimens, so the possibility that it does not detect novel virus 16
strains or species cannot be ruled out (27). Furthermore, arenaviruses are known to have 17
highly genetic variability, and false negative problems occasionally arise with some 18
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particular primer sets for RT-PCR. On the other hand, the Ag-capture ELISAs 1
established in the present study recognized highly conserved epitopes, suggesting that 2
Ag-capture ELISA may be useful for the diagnosis of suspected patients. 3
In conclusion, we developed Ag-capture ELISA systems using newly produced MAbs 4
against JUNV-NP, and showed that JUNV antigen was detected specifically by the 5
Ag-capture ELISA using MAb C6-9. On the other hand, the antigens of all human 6
pathogenic South American arenaviruses could be detected by Ag-capture ELISA using 7
MAbs C11-12, or E-4-2. The combined use of these Ag-capture ELISAs in the present 8
study may be useful for the diagnosis and differentiation of viral HFs caused by South 9
American arenavirus infections. 10
11
Acknowledgments 12
We thank M. Ogata, I. Iizuka, and T. Shiota for their helpful assistance. This work was 13
supported in part by a grant-in-aid from the Ministry of Health, Labour and Welfare of 14
Japan and the Japan Society for the Promotion of Science.
15
16
17
18
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References 1
1. Bockstahler, L. E., P. G. Carney, G. Bushar, and J. L. Sagripanti. 1992. 2
Detection of Junin virus by the polymerase chain reaction. J Virol Methods 3
39:231-5. 4
2. Buchmeier, M. J., J. C. de la Torre, and C. J. Peters. 2007. Field's Virology 5
5ed, vol. 2. Lippincott Williams & Wilkins. 6
3. Bui, H. H., J. Botten, N. Fusseder, V. Pasquetto, B. Mothe, M. J. Buchmeier, 7
and A. Sette. 2007. Protein sequence database for pathogenic arenaviruses. 8
Immunome Res 3:1. 9
4. Charrel, R. N., and X. de Lamballerie. 2003. Arenaviruses other than Lassa 10
virus. Antiviral Res 57:89-100. 11
5. Delgado, S., B. R. Erickson, R. Agudo, P. J. Blair, E. Vallejo, C. G. Albarino, 12
J. Vargas, J. A. Comer, P. E. Rollin, T. G. Ksiazek, J. G. Olson, and S. T. 13
Nichol. 2008. Chapare virus, a newly discovered arenavirus isolated from a fatal 14
hemorrhagic fever case in Bolivia. PLoS Pathog 4:e1000047. 15
6. Enria, D. A., A. M. Briggiler, and Z. Sanchez. 2008. Treatment of Argentine 16
hemorrhagic fever. Antiviral Res 78:132-9. 17
7. Garcia Franco, S., A. M. Ambrosio, M. R. Feuillade, and J. I. Maiztegui. 18
on March 19, 2021 by guest
http://cvi.asm.org/
Dow
nloaded from
23
1988. Evaluation of an enzyme-linked immunosorbent assay for quantitation of 1
antibodies to Junin virus in human sera. J Virol Methods 19:299-305. 2
8. Gonzalez, J. P., M. D. Bowen, S. T. Nichol, and R. Rico-Hesse. 1996. Genetic 3
characterization and phylogeny of Sabia virus, an emergent pathogen in Brazil. 4
Virology 221:318-24. 5
9. Ikegami, T., M. Niikura, M. Saijo, M. E. Miranda, A. B. Calaor, M. 6
Hernandez, L. P. Acosta, D. L. Manalo, I. Kurane, Y. Yoshikawa, and S. 7
Morikawa. 2003. Antigen capture enzyme-linked immunosorbent assay for 8
specific detection of Reston Ebola virus nucleoprotein. Clin Diagn Lab Immunol 9
10:552-7. 10
10. Kitts, P. A., and R. D. Possee. 1993. A method for producing recombinant 11
baculovirus expression vectors at high frequency. Biotechniques 14:810-7. 12
11. Lozano, M. E., D. Enria, J. I. Maiztegui, O. Grau, and V. Romanowski. 1995. 13
Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase 14
PCR-based assay. J Clin Microbiol 33:1327-32. 15
12. Lozano, M. E., P. D. Ghiringhelli, V. Romanowski, and O. Grau. 1993. A 16
simple nucleic acid amplification assay for the rapid detection of Junin virus in 17
whole blood samples. Virus Res 27:37-53. 18
on March 19, 2021 by guest
http://cvi.asm.org/
Dow
nloaded from
24
13. Maiztegui, J., M. Feuillade, and A. Briggiler. 1986. Progressive extension of 1
the endemic area and changing incidence of Argentine Hemorrhagic Fever. Med 2
Microbiol Immunol 175:149-52. 3
14. Matsuura, Y., R. D. Possee, and D. H. Bishop. 1986. Expression of the 4
S-coded genes of lymphocytic choriomeningitis arenavirus using a baculovirus 5
vector. J Gen Virol 67 1515-29. 6
15. Meyer, B. J., J. C. de la Torre, and P. J. Southern. 2002. Arenaviruses: 7
genomic RNAs, transcription, and replication. Curr Top Microbiol Immunol 8
262:139-57. 9
16. Mills, J. N., B. A. Ellis, K. T. McKee, Jr., T. G. Ksiazek, J. G. Oro, J. I. 10
Maiztegui, G. E. Calderon, C. J. Peters, and J. E. Childs. 1991. Junin virus 11
activity in rodents from endemic and nonendemic loci in central Argentina. Am J 12
Trop Med Hyg 44:589-97. 13
17. Morales, M. A., G. E. Calderon, L. M. Riera, A. M. Ambrosio, D. A. Enria, 14
and M. S. Sabattini. 2002. Evaluation of an enzyme-linked immunosorbent 15
assay for detection of antibodies to Junin virus in rodents. J Virol Methods 16
103:57-66. 17
18. Niikura, M., T. Ikegami, M. Saijo, I. Kurane, M. E. Miranda, and S. 18
on March 19, 2021 by guest
http://cvi.asm.org/
Dow
nloaded from
25
Morikawa. 2001. Detection of Ebola viral antigen by enzyme-linked 1
immunosorbent assay using a novel monoclonal antibody to nucleoprotein. J 2
Clin Microbiol 39:3267-71. 3
19. Riera, L. M., M. R. Feuillade, M. C. Saavedra, and A. M. Ambrosio. 1997. 4
Evaluation of an enzyme immunosorbent assay for the diagnosis of Argentine 5
haemorrhagic fever. Acta Virol 41:305-10. 6
20. Saijo, M., M. C. Georges-Courbot, P. Marianneau, V. Romanowski, S. 7
Fukushi, T. Mizutani, A. J. Georges, T. Kurata, I. Kurane, and S. Morikawa. 8
2007. Development of recombinant nucleoprotein-based diagnostic systems for 9
Lassa fever. Clin Vaccine Immunol 14:1182-9. 10
21. Saijo, M., M. Niikura, S. Morikawa, T. G. Ksiazek, R. F. Meyer, C. J. Peters, 11
and I. Kurane. 2001. Enzyme-linked immunosorbent assays for detection of 12
antibodies to Ebola and Marburg viruses using recombinant nucleoproteins. J 13
Clin Microbiol 39:1-7. 14
22. Saijo, M., T. Qing, M. Niikura, A. Maeda, T. Ikegami, C. Prehaud, I. 15
Kurane, and S. Morikawa. 2002. Recombinant nucleoprotein-based 16
enzyme-linked immunosorbent assay for detection of immunoglobulin G 17
antibodies to Crimean-Congo hemorrhagic fever virus. J Clin Microbiol 18
on March 19, 2021 by guest
http://cvi.asm.org/
Dow
nloaded from
26
40:1587-91. 1
23. Saijo, M., T. Qing, M. Niikura, A. Maeda, T. Ikegami, K. Sakai, C. Prehaud, 2
I. Kurane, and S. Morikawa. 2002. Immunofluorescence technique using 3
HeLa cells expressing recombinant nucleoprotein for detection of 4
immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus. J 5
Clin Microbiol 40:372-5. 6
24. Saijo, M., Q. Tang, B. Shimayi, L. Han, Y. Zhang, M. Asiguma, D. Tianshu, 7
A. Maeda, I. Kurane, and S. Morikawa. 2005. Antigen-capture enzyme-linked 8
immunosorbent assay for the diagnosis of crimean-congo hemorrhagic fever 9
using a novel monoclonal antibody. J Med Virol 77:83-8. 10
25. Sanchez, A., D. Y. Pifat, R. H. Kenyon, C. J. Peters, J. B. McCormick, and 11
M. P. Kiley. 1989. Junin virus monoclonal antibodies: characterization and 12
cross-reactivity with other arenaviruses. J Gen Virol 70 ( Pt 5):1125-32. 13
26. Ure, A. E., P. D. Ghiringhelli, R. D. Possee, S. Morikawa, and V. 14
Romanowski. 2008. Argentine hemorrhagic fever diagnostic test based on 15
recombinant Junin virus N protein. J Med Virol 80:2127-33. 16
27. Vieth, S., C. Drosten, R. Charrel, H. Feldmann, and S. Gunther. 2005. 17
Establishment of conventional and fluorescence resonance energy transfer-based 18
on March 19, 2021 by guest
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nloaded from
27
real-time PCR assays for detection of pathogenic New World arenaviruses. J 1
Clin Virol 32:229-35. 2
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Figure legends 1
Fig. 1. Reactivity of each monoclonal antibody with rNPs of arenaviruses. Each 2
purified rNP (100 ng/well) was coated onto the microplates as described in the text, and 3
the reactivity of each MAb to the rNP of JUNV (black box), MACV (dark gray box), 4
GTOV (light gray box), SABV (shaded box), CHPV (white box), and LASV (lined box) 5
was measured. The MAbs are shown on the x axis. Results indicate the OD value at 405 6
nm. 7
8
Fig. 2. Reactivity of each monoclonal antibody in antigen-capture ELISA. Purified 9
MAbs were coated onto the microplates as described in the text, and their ability to 10
capture the rNP of JUNV (filled circle), MACV (filled triangle), GTOV (open triangle), 11
SABV (filled diamond), CHPV (open diamond), and LASV (filled square) was 12
examined at various concentrations in the Ag-capture format. Results indicate the OD 13
value at 405 nm. 14
15
Fig. 3. Reactivity of MAbs C6-9, C11-12, and E4-2 with GST-tagged JUNV-NP by 16
Western blotting. Schematic drawings of polypeptides of JUNV-NP are shown on the 17
left of the figure and the aa positions of each polypeptide are indicated on the left. The 18
reactivity of MAbs and control MAb against GST to these polypeptides by Western 19
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blotting are shown on the right of the figure. The asterisks indicate the reacted 1
polypeptides. 2
3
Fig. 4. Determination of the epitope on JUNV-NP recognized by MAbs C6-9 (A), 4
C11-12 (B), and E4-2 (C). (A, B) The ability of synthetic deca-peptides to block the 5
reactivity of MAbs to JUNV-rNP was examined by ELISA. The aa sequences and 6
positions of synthetic peptides used in the assay are shown on the y axis. The synthetic 7
peptides at aa 1-10, and 97-106 are used as negative control peptides for MAb C6-9, 8
and C11-12, respectively. Results indicate the OD value at 405 nm. MAb C6-9 was 9
confirmed to react with 8 aa residues (PPSLLFLF) positioned from 551 to 558, as 10
represented by the shaded box (A). Similarly, MAb C11-12 was confirmed to react with 11
6 aa residues (WTQSLR) positioned from 12 to 17 (B). (C) The reactivity of MAb E4-2 12
with GST-tagged partial polypeptides of the JUNV-NP was examined by ELISA. 13
Schematic drawings of polypeptides of JUNV-NP are shown on the left of the figure and 14
the aa positions of each polypeptide are indicated on the y axis. NC represented GST 15
protein without any JUNV-NP sequences. Reactivity of MAb E4-2 to each partial 16
JUNV-NP is indicated by the adjusted OD405, which was calculated by dividing the 17
OD405 of MAb E4-2 by that of the anti-GST MAb to the corresponding antigens. MAb 18
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E4-2 was confirmed to react with 8 aa residues (KEVDRLMS) positioned from 72 to 79, 1
as indicated at the bottom of the figure. 2
3
Fig. 5. Schematic representation of epitopes of JUNV-NP recognized by monoclonal 4
antibodies. The amino acd (aa) sequences of the epitopes of JUNV-NP recognized by 5
MAbs C6-9, C11-12, and E4-2 are shown in boldface and the aa positions are shown 6
above the sequence. The aa sequences of the epitopes are compared to those of MACV, 7
GTOV, SABV, and CHPV. The aa residues different from the JUNV-NP are underlined. 8
Because the aa sequence of the corresponding region is conserved among strains in each 9
virus species, a single sequence is represented in the figure for each virus species. The 10
GenBank accession numbers for the S genes of JUNV are NC_005081, DQ272266, 11
AY746353, AY619641, AY358023, D10072, U70802, U70803, and U70804. Those for 12
the S genes of MACV are NC_005078, AY924208, AY924207, AY924206, AY924205, 13
AY924204, AY924203, AY924202, AY571959, AY624355, AY619645, AY571904, 14
AF485260, AY129248. Those for GTOV are NC_005077, AY497548, AF485258, 15
AY129247. Those for SABV and CHPV are NC_006317 and NC_010562, respectively. 16
17
18
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Table 1. Reactivity of the Mabs with NPs of arenaviruses in IFA
Reactivity of MAb* with NP of:
MAb
JUNV MACV GTOV SABV CHPV LASV
C6-9 + - - - - -
C11-12 + + + + + -
E4-2 + - - - - -
* “+” and “-” indicate positive and negative reactions, respectively.
The expression of each NP in Hela cells was confirmed by IFA using rabbit polyclonal
antiboby produced against each NP.
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2 531 . 522 . 5ue (
405 n
m)
JUNV
MACV
GTOV0 . 5 1
OD
valu
SABV
CHPV
LASV0C6-9 C11-12 E4-2
F i g ." 1
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C6ど 9
0.8 1
1.2
JU
NV
MA
CV
GT
OV
n m )
C
0
0.2
0.4
0.6
0.8
0
0
5
5
5
3
2
1
6
8
9
5
SA
BV
CH
PV
LA
SV
O D" v a lu e " (4 0 5
"
500
250
125
62.5
31.25
15.63
7.82
3.91
1.96
0.98
0.49
0.25
rNP" (ng/well)C11ど 123
5 4JU
NV
D05 1
1.5 2
2.5 3
3.5
MA
CV
GT
OV
SA
BV
CH
PV
LA
SV
O D" v a l u e " (4 0 5
" n m )0
0.5
500
250
125
62.5
31.25
15.63
7.82
3.91
1.96
0.98
0.49
0.25
rNP" (ng/well)E4ど 2E1
.5 2
2.5 3
3.5 4
JU
NV
MA
CV
GT
OV
SA
BV
CH
PV
LA
SV
v a lu e" (4 0 5" n m )
Fig." 20
0.5 1
500
250
125
62.5
31.25
15.63
7.82
3.91
1.96
0.98
0.49
0.25LA
SV
rNP" (ng/well)O D
" v
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1-5641-235
JUNV NP 25 35 50 75 25 35 50 75 25 35 50 75 25 35 50 75* * * (kDa)*1 235384-5641-5954-117465-531524-564
* * * ***** * *GST
GST C6-9 C11-12 E4-2
** *F i g .
" 3
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0 0 . 5 1 1 . 5 2 2 . 5 3 0 0 . 5 1 1 . 5 2C D
543 EEELTPLLPP 552
544 EELTPLLPPS 553
545 ELTPLLPPSL 554
546 LTPLLPPSLL 555
547 TPLLPPSLLF 556
5 KEVPSFRWTQ 14
6 EVPSFRWTQS 15
7 VPSFRWTQSL 16
8 PSFRWTQSLR 17
9 SFRWTQSLRR 18
548 PLLPPSLLFL 557
549 LLPPSLLFLP 558
550 LPPSLLFLPK 559
551 PPSLLFLPKA 560
552 PSLLFLPKAA 561
553 SLLFLPKAAY 562
10 FRWTQSLRRG 19
11 RWTQSLRRGL 20
12 WTQSLRRGLS 21
13 TQSLRRGLSQ 22
14 QSLRRGLSQF 23
15 SLRRGLSQFT 24553 SLLFLPKAAY 562
554 LLFLPKAAYA 563
555 LFLPKAAYAL 564
1 MAHSKEVPSF 10
15 SLRRGLSQFT 24
16 LRRGLSQFTQ 25
17 RRGLSQFTQT 26
97 DELMELASDL 1060 0 . 5 1 1 . 5 2 2 . 5 31ど
5 6 46 7ど
5 6 47 2ど
5 6 4E 7 2ど
5 6 47 3ど
5 6 47 4ど
5 6 47 5ど
5 6 47 7ど
5 6 41ど
7 61ど
7 71ど
7 81ど
7 9 F i g ."
41ど
8 0N C72 KEVDRLMS 79
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LWPX"PR
C6-9C11-12 E4-2
JUNV: WTQSLR
MACV: WTQSLR
34 39KEVDRLMSMRSV
KEVDRLMSMKSI
94 9;PPSLLFLP
PLSLLFLP
77:7731:2 :5
GTOV: WTQSLR
SABV: WTQSLR
CHPV: WTQSLR
KEVDRLMSMKSV
KEVDNLMSMKSS
REVDNLMAMKSA
PNTLVFQA
PDALLFTL
PEVLLFTM
Minimal epitope
F i g ." 5
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